Of the mechanisms bacteria use to evade the effects of aminoglycoside (AG) antibiotics, enzymatic modification has the most clinical relevance due to the promiscuous nature of the DNA encoding the genes for these enzymes. One such enzyme, aminoglycoside 3â€™-phosphotransferase IIa (APH(3â€™)-IIa), is used as a model for understanding this modification at a molecular level along with anticipating the evolution of AG resistance. To study the structure-function relationships of this enzyme, we previously determined the crystal structure of APH(3â€™)-IIa and modeled its ATP binding site. We identified a lysine that appeared to be involved in the binding of ATP and generated a conserved lysine to arginine mutant to better assess this residueâ€™s functional contributions. We determined the binding kinetics for ATP to this mutant and employed active-site labeling using an azido ATP analog to confirm the importance of this conserved lysine. Competition experiments with tryptophan during activesite labeling revealed that a conserved tryptophan residue in the amino-terminus of the enzyme may be involved directly or by association with ATP binding. Together, these data give us a clearer picture of how ATP associates with APH(3â€™)-II and could aid in the development of small chemical compounds which specifically inhibit its activity.